Metallo-organic cobalt compounds and uses thereof

ABSTRACT

The present invention provides a pharmaceutical composition of a complex compound having the structure: ##STR1## wherein R 1  and R 1 , are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group; 
     wherein R 2  and R 2 , are the same or different and each is hydrogen, an unbranched alkyl group, a halide or a group having the structure ##STR2## wherein R is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R 3  and R 3 , are the same or different and each is hydrogen or an alkyl group; 
     wherein X and X&#39; are the same or different and each is a water soluble group having weak to intermediate ligand field strength; and 
     Q is a soluble, pharmaceutically acceptable negative ion.

This is a continuation of application Ser. No. 07/895,526, filed Jun. 2,1992, now U.S. Pat. No. 5,258,403, which, in ram, is a division ofapplication Ser. No. 07/606,070, filed Oct. 30, 1990, now U.S. Pat. No.5,142,076, which, in turn, is a continuation of U.S. patent applicationSer. No. 07/279,417, filed Dec. 2, 1988, now, U.S. Pat. No. 5,049,557which, in turn, is a continuation-in-part of application Ser. No.07/147,713, and application Ser. No. 07/147,714, both filed Jan. 25,1988, now U.S. Pat. Nos. 4,866,054 and 4,866,053, respectively, which,in turn are continuation-in-parts of application Ser. No. 06/862,804,filed May 13, 1986, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to metallo-organic cobalt compounds andtheir use in the treatment of subjects for inflammatory conditions,particularly arthritis, and like .conditions caused by oxygen freeradicals. The invention also relates to a method of treating burns andwounds and the use of the subject compounds as antimicrobial agents.

It has been recognized for some time that inflammation in mammalianspecies can be traced at least in part to active oxygen species,including superoxide, and radicals associated therewith at theinflammatory site. Considerable research has been undertaken to measureand detect oxygen radicals, to establish the mechanisms whereby enzymessuch as superoxide dismutase are effective in countering oxygen radicaltoxicity, and in the development and use of copper amine oxidases inpreventing tissue damage and even in promoting damage-tissue recovery.Recently superoxide dismutase, which decomposes highly toxic oxygen freeradicals, has been put into veterinary use as an anti-inflammatory agentwith efficacy in the treatment of conditions such as traumatic arthritisof horses.

Free radical toxicity has also been identified as operative inpoisonings by various pharmacologic agents. Enzymes such as superoxidedismutase have been used as antidotes to nullify the toxic effects ofthe putative free radical generators in vivo.

However, the compounds which have been developed heretofore foractive-oxygen or superoxide antagonism and destruction in vivo have notproven as effective as desired, or are characterized by side reactions,or cannot be made in commercially significant quantities at reasonablecost.

The present invention provides compounds and methods for the treatmentof subjects having inflammatory conditions or other conditionsassociated with free radicals whereby the above-mentioned drawbacks areeliminated. The invention provides a method for treating a subjecthaving a condition resulting from active oxygen or superoxide toxicitywhich may result in acute or chronic inflammation or other disorders,such as arthritis.

The invention also provides a method of treating burns and wounds in asubject. The treating of burns of the first, second, and third degreehas long been and remains one of the most difficult medical problems.The criteria for success of any method for treating a burn includesproper contraction of the wound, epithelialization, hair folliclepreservation, and the assessment of newly formed granulation tissue.Contraction represents the difference between the initial wound size ofthe burn and the size of the burn twelve days later (12th post burn dayor PBD), which includes both open and healed areas calculated as apercentage of the initial wound size.

Epithelialization represents the percentage of the newly covered areasof the burn surface on the 12th PBD out of the total wound area on thatsame day. The presence of hair follicles indicates maintenance orrestoration of dermal microcirculation and prevention of tissue ischemiaand thus ischemic and postischemic damage. The preservation of hairfollicles and their count should be carried out microscopically intissue sections. Also important in the evaluation of medicament oftreating burns is the assessment of newly formed granulation tissue. Thethickness of the new collagen layer synthesized in the healing burnshould be measured on PBD 12.

As part of the overall management of burn wounds, a topicallyantibacterial agent, such as silver sulfadiazine, may be applied.Unexpectedly, it has been found that the compounds of the presentinvention may be used as an antibacterial agent which can help preventthe colonization of the wound by pathologic agents. The method of thepresent invention maximizes epithelialization of the burn on amacroscopic level and maximizes hair follicle preservation on amicroscopic level.

Additionally, it has been unexpectedly found that the compounds of thepresent invention cause regression in the growth of tumor cells in vivo.A method is provided for the treatment of a subject afflicted with atumor Which comprises administering to the subject the compounds of thepresent invention in an amount sufficient to cause regression of thetumor cells.

SUMMARY OF THE INVENTION

The present invention provides a complex compound having the structure:##STR3## wherein R₁ and R₁, are the same or different and each is analkyl group, a phenyl group or a substituted derivative of a phenylgroup;

wherein R₂ and R₂, are the same or different and each is hydrogen, anunbranched alkyl group, a halide or a group having the structure##STR4## wherein R is hydrogen, an alkoxide group, an alkyl group, orOH; wherein R₃ and R₃, are the same or different and each is hydrogen oran alkyl group; and

wherein X and X' are the same or different and each is a water solublegroup having weak to intermediate ligand field strength.

The invention also provides a complex compound having the structure:##STR5## wherein R₁ and R₁, are the same or different and each is analkyl group, a phenyl group or a substituted derivative of a phenylgroup;

wherein R₂ and R₂, are the same or different and each is an unbranchedalkyl, group, a halide or a group having the structure ##STR6## whereinR is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R₃ andR₃, are the same or different and each is hydrogen or an alkyl group;

wherein X and X' are the same or different and each is a water solublegroup having weak to intermediate ligand field strength; and

Q is a soluble, pharmaceutically acceptable negative ion.

The invention also provides a method of treating a subject having acondition associated with the presence of free radicals in quantitiessufficient to cause undesirable symptoms. A method of treating a woundor a burn and a method for treating a subject afflicted with tumor cellsso as to cause regression of the tumor cells are also provided. Themethods of the present invention involve administering to the subject(or topically administering to the burn or wound) a compound having thestructure: ##STR7## wherein R₁ and R₁, are the same or different andeach is an alkyl group, a phenyl group or a substituted derivative of aphenyl group;

wherein R₂ and R₂, are the same or different and each is hydrogen, anunbranched alkyl group, a halide or a group having the structure##STR8## wherein R is hydrogen, an alkoxide group, an alkyl group, orOH; wherein R₃ and R₃, are the same or different and each is hydrogen oran alkyl group;

wherein X and X' are the same or different and each is a water solublegroup having weak to intermediate ligand field strength; and

Q⁻ is a soluble, pharmaceutically acceptable negative ion.

Furthermore the invention provides pharmaceutical compositions for thetreatment of tumor cells in a subject or for the treating of undesirablesymptoms associated with the presence of free radicals. The compositionscomprise an effective amount of the compound and a pharmaceuticallyacceptable carrier. Also provided is an antimicrobial composition whichcomprises a suitable carrier and the compound in an amount effective tosuppress the growth of microorganisms.

BRIEF DESCRIPTION OF THE FIGURES:

FIG. 1: A Diagram of the Molecular Orbitals for the Cobalt Complex.

FIG. 2(a) and 3(a): Graph of Body Weight: vs. Days for Anti-ascitesActivity of Compound 23.

FIG. 2(b) and 3(b): Graph of % Viability vs. Days for Anti-ascitesActivity of Compound 23.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a complex compound having the structure:##STR9## wherein R₁ and R₁, are the same or different and each is analkyl group, a phenyl group or a substituted derivative of a phenylgroup;

wherein R₂ and R₂, are the same or different and each is hydrogen, anunbranched alkyl group, a halide or a group having the structure##STR10## wherein R is hydrogen, an alkoxide group, an alkyl group, orOH; wherein R₃ and R₃, are the same or different and each is hydrogen oran alkyl group; and

wherein X and X' are the same or different and each is a water solublegroup having weak to intermediate ligand field strength.

Also provided is a complex compound having the structure: ##STR11##wherein R₁ and R₁, are the same or different and each is an alkyl group,a phenyl group or a substituted derivative of a phenyl group;

wherein R₂ and R₂, are the same or different and each is an unbranchedalkyl group, a halide or a group having the structure ##STR12## whereinR is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R₃ andR₃, are the same or different and each is hydrogen or alkyl group;

wherein X and X' are the same or different and each is a water solublegroup having weak to intermediate ligand field strength; and

Q is a soluble, pharmaceutically acceptable negative ion.

The compounds of the present invention may be crystallized with numerouscounteranions. Those which are pharmaceutically acceptable and are watersoluble, such as halide ions, PF₆ ⁻ and BF₄ ⁻, are preferred. Thebromine salts of the present compounds are the most preferred becausethey are the easiest to crystallize and are more water soluble thanother salts of the compounds.

As discussed above the R₁ and R₁, groups of the compound may be the sameor different from each other and each may be an alkyl group, a phenylgroup or a substituted derivative of a phenyl group. Preferably thealkyl group is a C₁ -C₅ group with methyl, ethyl and butyl groups beingparticularly preferred. Suitable substituted derivatives of the phenylgroup are derivatives wherein each substituent is a halide, an alkylgroup or a group having the structure ##STR13## where R is hydrogen, analkoxide group, an alkyl group or an OH group. To date the most usefulderivatives have proven to be those in which the substituents arehalides, carbonyl groups or alkyl groups.

The R₂ and R₂, groups of the complex compounds of the present inventionmay also be the same or different and may be hydrogen, an unbranchedalkyl group, a halide or a group having the structure ##STR14## where Ris hydrogen, an alkoxide group, an alkyl group or an OH group. Incertain embodiments, it is preferred that R₂ and R₂, are chlorine orhydrogen atoms or a C₁ -C₃ alkyl group. In embodiments where R₂ has astructure ##STR15## it is preferred that R is hydrogen, a methyl groupor an OH group.

The R₃ and R₃, of the complex compounds of the present invention are thesame or different and each may be hydrogen or an alkyl group, preferablya C₁ -C₃ alkyl group.

With respect to the X and X' groups, it is preferred that these groupsare water-soluble and have a weak to intermediate ligand field strength.Ligands are arranged in a spectrochemical series according to themagnitude of their field strength or the "Δ₀ " they bring about. Thesymbol Δ₀ represents the difference between the energies of the d_(xy),d_(xz), and d_(yz) orbitals and the d_(z) ² and d_(x) ² _(-y) ²orbitals. From experimental studies, it is known that the order ofligands, based on their ligand field strength, is approximately the samefor the complexes of all the transition metals in their common oxidationstates with only an occasional inversion of order between ligands thatstand near to one another. A typical order of some common ligands is asfollows:

    I.sup.- <Br.sup.- <Cl.sup.- <OH.sup.- <C.sub.2 O.sub.4.sup.2- <H.sub.2 O<NH.sub.3 <en<NO.sub.2.sup.- <CN.sup.-

The cyanide ion, which stands at the opposite end of the series from thehellions, has the strongest ligand field strength and induces thelargest d-orbital splitting of any ligand listed. On the other hand, thehalide ions such as Br⁻ and Cl⁻ have weak ligand field strength andinduces the smallest d-orbital splitting. Ligands such as NH₃ and OH⁻have-intermediate ligand field strength. For general backgroundinformation, see F. Albert Cotton and Geoffrey Wilkinson, "AdvancedInorganic Chemistry", John Wiley & Sons 4th ed, p. 663. In the presentinvention,it is preferred that X and X' are ligands with weak tointermediate ligand field strength, such as halides or NH₃, H₂ O, ordimethyl sulfoxide.

Particular preferred embodiments of the complex compounds of the presentinvention are as follows: ##STR16## wherein Ph is a phenyl group,

The invention also provides a pharmaceutical composition and method fortreating a subject having a condition associated with the presence offree radicals in quantities sufficient to cause undesirable symptoms,The pharmaceutical composition comprises a pharmaceutically acceptablecarrier and the compound of the present invention in an amount effectiveto alleviate the undesirable symptoms associated with the presence ofthe free radicals. The method comprises administering to the subject thecompound in an amount effective to alleviate the undesirable symptoms.

The method may be used for the treatment of any condition associatedwith free radicals. It is however most effective against conditionsassociated with oxygen free radicals, such as O₂ ⁻. Such conditions maycomprise inflammation, including synovial inflammation and arthritis,ulcers such as diabetic ulcers, and conditions caused by the loss ofcirculation due to free radicals, such as hair loss due to the loss ofdermal microcirculation.

Preferably, the method is used in the treatment of acute or chronicarthritis with a dosage of the compounds of 0.1 to 250 mg/kg (especiallyabout 1 to about 200 mg/kg) of body weight, but in all cases at most 50%of the LD₅₀ value of the compound, when the compound is administeredorally as is preferred. However, the complex compound or a combinationof the complexes can be administered subcutaneously or even topically ina suitable vehicle, e.g. physiological saline in the case of s.c.administration and dimethylsulfoxide (DMSO) in the case of a topicaladministration, although ointments, salves or like conventional vehiclesmay be employed.

For oral administration, the complex or mixture of complexes may beprepared in suitable dosage forms. For example it may be prepared asdragees, as capsules, as tablets, as an elixir or other oral dosageform.

The dose may be administered one to six times daily, depending upon theseverity of the inflammatory condition, preferably under medicalsupervision so that the dosage can be reduced or the number of dailyadministrations limited as the inflammatory condition subsides.

The compounds of the invention may also have prophylactic properties inpreventing the spread of arthritic inflammation and have been found tobe effective in reducing the severity of the actual condition whichdevelops in subjects who are prone to such inflammatory states. Thecompounds may also be effective in preventing postischemic heart damageand for geriatric applications other than antiarthritics.

It is also contemplated that the compounds of the invention may be usedin conjunction with known anti-inflammatory agents with propionic acidside chains, especially indomethacine, to further alleviate thesuffering of arthritic inflammation.

The invention also provides an antimicrobial composition which comprisesa suitable carrier and the compound of the present invention in anamount effective to suppress the growth of microorganisms. Theantimicrobial composition or compounds of the present invention may beused for the treatment of a wound or burn by topically administering tothe wound or burn the composition or compounds. The compounds may alsobe used in the treatment of conditions which are normally treated withantimicrobial or antibacterial agents. For example, the compounds may beused in the topically treatment of an infectious disease or an abrasionas an antibiotic.

The cobalt complexes of the present invention are water-soluble and maybe dissolved in a number of carriers. Suitable carriers include polar,protic solvents such as water or especially normal saline. The cobaltcomplexes may also be suspended in a suspension medium that is notmiscible with water, for example petrolatum.

The concentration of the complexes in the solvent or suspension mediumcan vary from 0.1 to 50 mg/ml. A preferred concentration range liesbetween 1 and 10 mg/ml.

The complexes may be applied to the site of the burn, wound, abrasion,etc. in the form of an aerosol, in the form of a salve, ointment, orcream, or directly in a liquid solvent, preferably normal saline, by theuse of a medicine dropper. Furthermore the complexes may be applied tothe burn site together with a topical anaesthetic agent such asbenzocaine, a soothing agent such as menthol, an antibacterial agentsuch as bacitracin, or a combination of these ingredients.

The method is particularly effective for killing microorganisms such asStrep. β hemolytic, Strep. α hemolytic, Enterococci, Staph. coagulase(+), Staph. coagulase (-), E. Coli, Klebsiella, Pseudomonas, Proteus, orC. albicans.

It has also been-found that the compounds of the present invention areeffective in the treatment of subjects having tumors. Administration ofthe compounds to a subject afflicted with tumor cells causes aregression in the growth of the tumor cells. The compound may bedirectly administered to the subject or it may be administered in apharmaceutical composition which comprises an e effective anti-tumoramount of the compound and a pharmaceutically acceptable carrier. Thismethod has been found to be particularly effective in the treatment oftumors associated with ascites cells.

It is also contemplated that the compounds and compositions of thepresent invention may be used in the treatment of other conditionsassociated with free radicals, such as poisonings with pharmacologicagents or conditions caused by ionizing radiation. The compounds mayalso be used in industrial applications where free radicals, such asoxygen radicals, are undesirable; for example, in wastewater treatmentas an oxygen scavenger.

Other embodiments and uses for the compounds of the present inventionwill become apparent to those skilled in the art upon a reading of thepresent disclosure. These uses and embodiments are intended to be withinthe spirit and scope of the present invention.

The invention is further illustrated in the Experimental Details sectionwhich follows. This section is set forth to aid in an understanding ofthe invention but is not intended to, and should not be construed to,limit in any way the invention as set forth in the claims which followthereafter.

EXPERIMENT DETAILS

I. Synthesis:

Several cobalt(III) complex compounds having the general formula[CoL(NH₃)₂ ]⁺, wherein L represents a tetradentate ligand with twooxygen and two nitrogen donor atoms, have been prepared andcharacterized. The purity of the compounds was established by elementalanalysis, N.M.R. and U.V.-visible spectroscopy. The geometry around thecobalt atom was established by X-ray crystallography. Analytical reagentgrade chemicals were used without further purification. A detaileddescription for a typical preparation for each of the compounds follows.Of the compounds described only [Co(L23) (NH₃)₂ ]⁺ have been reportedpreviously(¹).

1. Synthesis of [CoL23(NH₃)₂ ]Cl,(L23=N,N'-bis(acetylacetone)ethylenediimine) ##STR17##

The tetradentate ligand was prepared as described by McCarthy.sup.(2).To 0.023 moles of L23 dissolved in 100 ml of methanol was added 0.016moles of COCl₂ H₂ O dissolved in 100 ml of methanol. Concentrated NH₄ OHwas added dropwise with continuous stirring until the pH became basic(pH=8). Stirring was continued for an additional three hours. Theresulting precipitate was filtered and recrystallized from hotethanol/water.

Found; C, 40.9%; H. 6.75%; N, 16.1%. Calculated for C₁₂ H₂₄ O₂ N₄ CoCl;C, 41.09%; H, 6.90%; N, 15.9%. MW=350.4.

2. Synthesis of [Co(L64)(NH₃)₂ ]Cl, (L64=N,N'-bis(benzoylacetoneethylenediimine). ##STR18##

L64 was prepared as described by McCarthy.sup.(2). To 0.022 moles of L64dissolved in 100 ml of CH₂ Cl₂ was added a filtered solution of 0.020moles of CoCl₂ 6H₂ O in 100 ml of absolute methanol. A concentratedsolution of NH₄ OH was added slowly with continuous stirring until thesolution became basic (pH=8). Stirring continued for an additional 4hrs. The solution was concentrated to a volume of 50 ml and filtered.Slow evaporation of the mother liquor gave the desired product.

Calculated for C₂₂ H₂₈ O₂ N₄ CoCl.H₂ O: C, 54.72%; H, 6.20%; N, 11.61%;Found: C, 54.61%; H, 6.05%; N, 11.49%. MW=482.4

3. Synthesis of [Co(L67)(NH₃)₂ ]Cl,(L67=N,N'-bis(chloroacetylacetone)ethylenediimine) ##STR19##

L67 was prepared as previously described.sup.(3). [Co(L67)(NH₃)₂ ]Cl wasprepared by the same procedure as [C0L23)(NH₃)₂ ]Cl. Found; C, 34.61%;H, 5.38%; N, 13.03%. Calculated for C₁₂ H₂₂ O₂ N₄ CoCl₃ ; C, 34.35%; H,5.02%; N, 13.35%. MW=419.32

4. Synthesis of [Co(L68)(NH₃)₂ Br,(L68=N,N'-bis(chlorobenzoylacetone)ethylenediimine) ##STR20##

L68 was prepared by direct chlorination of L64 with a slight excess ofN-chlorosuccinimide (NClS).sup.(4) . 0.1 moles of L64 was dissolved in700 ml of ice cold CH₂ CL₂. 0.25 moles of NC1S was added and thereaction stirred for 20 min. The ligand precipitated as a yellow powderwhich was filtered, washed with ether and dried. 0.005 moles of L68 wassuspended in 100 ml of absolute ethanol under nitrogen to which 0.01moles of KOH dissolved in 25 ml of methanol was added. 0.0045 moles ofCo(OAc)₂.4H2O in 25 ml of methanol was slowly added to the abovesuspension with continuous stirring. The reaction mixture was heated to50° C. for 1 hr. After cooling, the orange precipitate of [Co(L68) ] wasfiltered, washed with ethanol and dried. 0. 002 moles of the Co(II)complex [CoL68] was suspended in 100 ml of methanol under N₂. 1 ml of30% H₂ O₂ was added dropwise and anhydrous ammonia was bubbled throughthe reaction mixture until all the Co(II) complex had been dissolved. Atthis point, bubbling of N₂ was discontinued and the solution was stirredfor an additional 1 hr. The solution was, filtered and three equivalentsof NaBr dissolved in a minimum amount of water was added. Slowevaporation of the solution yielded the desired product.

Found: C, 43.44%; H, 4.46%; N, 9.07%. Calculated for C₂₂ H₂₆ BrCl₂ N₄ O₂Co. H₂ O: C, 43.58%; H, 4.65%; N, 9.24%. MW=605.78

5. Synthesis of [Co(L69) (NH₃)₂ ]Cl.(L69=N.N'-bis(methylacetylacetone)ethylenediimine) ##STR21##

L69 was prepared by condensation of anhydrous ethylenediamine withmethylacetylacetone.sup.(5). The complex is prepared by the sameprocedure as described for [Co(L23)(NH₃)₂ ]Cl.

Calculated for C₁₄ H₂₈ N₄ O₂ CoCl.H₂ O; C, 42.42%, H, 7.57, N, 14,14%.Found: C, 42.14%; H, 7.48%; N, 14.01%. MW=396.4

6. Synthesis of [Co(L70)(NH₃)₂ ]Br,(L70=N,N'-bis(methylbenzoylacetone)ethylenediimine) ##STR22##

L70 was prepared by condensation of ethylene diamine withmethylbenzoylacetone.sup.(6). The Co(III) complex was prepared via theCo(II) complex by the procedure described for [Co(L68 )NH₃)₂ ]⁺.

Found: C, 46.87%; H, 5.03%; N, 8.95%. Calculated for C₂₄ H₃₂ B₁.N₄ O₂Co:47.07H, 5.27%; H, 5.27%; N,, 9.15%, MW=611.9

All complexes described can be crystallized with other counteranions.Those anions which give the best solubility in water here chosen here.

The-reaction of Co(II) complexes with molecular oxygen has been studiedextensively.sup.(7,8). Normally, cobalt(II) forms 2:1 peroxo bridgedcomplexes in aqueous solutions.sup.(8). In recent years, a number ofCo(II) complexes have been reported to give 1:1 cobalt-oxygen adducts atroom temperature. These complexes usually contain ligands which whenbound to Co(II) give rise to a low spin planar geometry. Addition ofbase and O₂ to these complexes leads to the formation of octahedralcomplexes where the base and the O₂ occupy axial positions.sup.(9).

On the basis of measurements utilizing a variety of physical techniquesit is now a well accepted fact that the most accurate electronicstructure description for the Co:O₂ moiety is a Co,(III) ion bound to O₂⁻, where the actual amount of Co→>O₂ electron transfer depends on thenature of the ligand and the donor set .sup.(9,10). It has been shownthat electron transfer increases with increase of the ligand fieldstrength.sup.(7). This can be easily understood from the molecularorbital diagram depicted in FIG. 1. In FIG. 1, the donor atoms A definethe basal plane of the molecule, while B represents the; axial ligands.As the ligand field strength of the donor atoms around the cobaltincreases, the metal orbitals (especially d_(z) ² and d² -y² which are σorbitals) are raised in energy relative to the π orbitals of O₂ and moreelectronic charge is trans feted from the metal to the bound dioxygen,i.e., the O₂ molecule attains more Ohd 2⁻ character. Thus, consideringelectronic structural arguments only, one can conclude that a Co(III)complex with a set of six donor atoms (four A donors, and two B donors)having an intermediate ligand field strength could be effective inreacting with O₂ ⁻ by either forming a stable Co(III)-O₂ adduct (the O₂substitutes one of the B ligands), or by oxidizing O₂ ⁻, liberatingdioxygen and yielding a Co(II) complex. The relative strength of thedifferent ligands is well known from the spectrochemical series.However, it should be quite clear that a sterically unstrained ligandsystem which can easily accomodate both metal oxidation states [Co(II),Co(III)] with minimal reorganization of the geometry around the: metaland which does not lead to a high spin Co(II) complex should facilitatethe reaction.

Thus, on the basis of geometrical, steric and electronic requirements wesuggest that a Co(III) complex having a quadridentate ligand whichimposes planarity on the. octahendral basal plane should be a suitablecandidate for reaction with the O₂ ⁻ radical anion. The effectiveness ofthis reaction will depend on the nature of the quadridentate ligand, itsligand field strength and on the nature of the axial ligands B.

The complexes [CoL(NH₃)₂ ]⁺ fit nicely with the set of requirementsgiven above. First, the six donor atoms N₄ O₂ give rise to anintermediate ligand field. Secondly, the quadridentate ligand L whenbound to Co(III) gives rise to a 6,5,6 ring system where the sixmembered rings are unsaturated, thereby ensuring the planarity of thecomplex basal plane without steric strain, as has been determined fromX-ray crystal structure analysis. Thirdly, the ligand L when bound toCo(II), gives a low spin, planar complex.

It should be pointed out that the unsaturation of the six-membered ringsis important not only because it ensures the required geometry, but alsobecause it provides for an effective pathway for transmitting electroniceffects of different substituents to the cobalt center, therebyaffecting the relative energies of the metal d orbitals.

II. In vivo Inflammatory Studies:

The drugs used are prepared just before performing the experiment. Drugsare dissolved at a concentration of 10⁻² M (or 2×10⁻² M or 4×10⁻² M) inpyrogen free sterile saline. The dissolved drug is then filtered througha sterile and pyrogen-free 0.2 micron filter (Acrodisc, Gelman).

Procedure

8-12 CD-1 female mice (Charles River), age 2-5 months are numbered,weighed and distributed to 2-3 cages, 3-4 mice in each cage. 0.2 ml ofpyrogen-free saline or drug are injected subcutaneously in a randomizedorder.

Thirty minutes after injecting the drugs or the saline, the right paw ofeach mouse is injected with 25 microliters of 1% carrageenin (viscarintype, Marine Colloids) in pyrogen-free saline or with 5 microliters(0.227 U) of xanthineoxidase (Sigma).

1.5 hr to 2 hrs after injecting the inflammatory stimulus to the rightpaw, both paws of the animal are amputated at the knee joint andweighed. The uninjected left paw serves as an internal control for thedegree of swelling of the right paw in each animal.

In some experiments the :surface temperature of the right and left pawswere recorded also before the amputation was performed.

Calculations

The difference in mg between the weight of the right and left paw incontrol animals (injected with saline) represents 100% of the acuteinflammatory response. Concomitantly, the difference between paws of thedrug treated animals is calculated and compared to control. Results for[CoL23(NH₃)₂ ]Cl (designated as "23" in tables) are shown in Tables 1, 2and 3 which follows.

                  TABLE 1                                                         ______________________________________                                        Effect of several products on carrageenin paw                                 oedema in mice measured by paw weight.                                                              Paw Weight                                                     No. of  total  route of                                                                             average                                                                              %     % inhib-                            Product                                                                              expts.  mice   admin. mg/kg  activity                                                                            ition-                              ______________________________________                                        saline --      --     *S.C.  --     100%  0                                   3      4       14     *S.C.  25     63.1% 36.9%                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Effect of several products on xanthine-oxidase                                paw oedema measured by temperature reduction                                         No. of  total  route of                                                                             average                                                                              %     % inhib-                            Product                                                                              expts.  mice   admin. mg/kg  activity                                                                            ition-                              ______________________________________                                        saline --      --     *S.C.  --     100%  0                                   23     2       6      *S.C.  22.7    31%  69%                                 ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        LD.sub.50 and ED.sub.50 of various drugs on xanthine                          oxidase and carrageenin paw oedema in mice                                                   stimulus                                                                      :xanthine                                                                     oxidase   carrageenin                                                         measurement                                                                         :temp.      weight mg                                    Drug    LD.sub.50    ED.sub.50   ED.sub.50                                    mg/kg                mg/kg       mg/kg                                        ______________________________________                                        23      75           <38         25                                           ______________________________________                                    

Compound 64 ([Co(L64 )(NH₃)₂ ]Cl) was found to be as effective as ormore effective than compound 23 in corresponding tests. Both compoundswere found to be effective in reducing oedema upon oral administrationin force feeding with pills containing 8 mg of active ingredient andadministered in a quantity sufficient to provide an effective dose. Thepills were enterally coated (see below).

Example 1

The composition of tablets is as follows:

    ______________________________________                                        active ingredient (one or both                                                                    25.0       mg.                                            of compounds 23 or 64                                                         corn starch         97.0       mg.                                            polyvinyl pyrrolidone                                                                             175.0      mg.                                            magnesium stearate  3.0        mg.                                                                300.0      mg.                                            ______________________________________                                    

The active ingredient and the corn starch are wetted by an aqueouspolyvinyl pyrrolidone solution of approx. 15% w/v, followed bygranulation, and drying of the wet granules at about 40°-45° C. Thedried granulate is thoroughly mixed with magnesium stearate, and themixture so obtained is further processed by a tablet machine, equippedwith an appropriate pressing tool, to give tablets of 300 mg. weightcontaining 25 mg. of active ingredient. One manufacturing lot includes100 tablets.

Example 2

Dragees of the following composition are prepared:

    ______________________________________                                        active ingredient (one or both                                                                    50.0       mg.                                            of compounds 23 or 64)                                                        lactose             94.0       mg.                                            polyvinyl pyrrolidone                                                                             4.0        mg.                                            magnesium stearate  2.0        mg.                                                                150.0      mg.                                            ______________________________________                                    

Granulates are prepared according to Example 1, and from them drageekernels of 150 mg. weight are pressed. The dragee kernels are coatedwith a layer containing sugar and talc followed by coloring with anapproved food colorant and polishing with beeswax.

Example 3

25 mg. of active ingredient (one or both of compounds 23 or 64) aredissolved in 100 ml. of distilled water. The solution is filled into 500ampules. In this way ampules containing 2 ml. of a solution containing25 mg./ml. of active agent each ,are obtained. The contents of an ampuleare injected subcutaneously.

Example 4

Gelatin capsules of the following composition are prepared:

    ______________________________________                                        active ingredient (one or both                                                                    25.0       mg.                                            of compounds 23 or 64)                                                        maize starch        122.0      mg.                                            colloidal silica    3.0        mg.                                                                150.0      0 mg.                                          ______________________________________                                    

The ingredients are homogenized, and the homogenate is put into hardgelatine capsules. 1000 capsules of 150 mg. (filling) weight each,containing 25.0 mg. of active ingredient per capsule, make a lot.

Example 5

Pills of the active ingredient are enterally coated with a melt of 45parts of n-butyl stearate, 30 parts of carnauba wax and 25 parts ofstearic acid, all by weight, at a temperature of 75° C.

II. Superoxide Scavenging

Compounds 67, 68, and 69 ([Co(L67)(NH₃)₂ ]Cl; [Co(L68)(NH3)₂ ]Br;[Co(L69)(NH₃)₂ ]Cl, respectively) were tested spectrophtmerically for O₂quenching (reduction of NBT in the presence of xanthine and xanthineoxidase as the generating system of superoxide radicals) . Thesecompounds were also evaluated by quenching of O₂ produced by macrophagesin tissue culture. Macrophages are a source of superoxide radicals ininflammatory states.

The activity of these compounds were assessed and compared to compounds23 and 64 Which hereinabove are reported to be effective superoxideradical scavengers and effective in the treatment of rat adjuvantarthritis. Subsequently, these two compounds were also found to beeffective in burn wound healing. Table 4 summarizes the results obtainedwith compounds 67, 68, 69 as compared with 23 and 64.

As depicted in Table 4 compounds 23 and 67 have given similar resultsboth in the test tube and with macrophages.

50% O₂ scavenging was achieved at a concentration of about 10⁻⁵ Mwhereas Compounds 64 and 68 gave diverse results with the two methods:in the test tube the compounds were very effective, yielding 50% O₋₂quenching at concentrations of 2-8×10⁻⁶ M whereas in the macrophagesystem there was a decrease in superoxide scavenging ability to therange of 10⁻⁴ M. Compound 69 was not as effective in O₂ scavenging asjudged by either method. BSA was used as a chelating agent, in order todetermine the stability of each ligand with the metal in physiologicalfluids. Compound efficacy in the presence of BSA was as follows:23>67>69>64.

                  TABLE 4                                                         ______________________________________                                        The drug concentration [M] at which 50% superoxide quenching                  is achieved, as measured by spectrophotometric and tissue culture             methods. Also, the concentration of the drug at which 50% of                  protein synthesis is inhibited in cultured macrophages.                                                         50%                                         Com-  Test tube                   inhibition of                               pound determina- Macrophages      protein                                     #     tion(a)    -BSA(b)    +BSA(c) synthesis(d)                              ______________________________________                                        23    7.7 ± 0.5 ×                                                                     7.0 ± 0.8 ×                                                                     1 × 10.sup.-4                                                                   6.0 × 10.sup.-5                           10.sup.-5  10.sup.-5                                                    64    8.0 ± 0.64 ×                                                                    1.4 ± 0.23 ×                                                                    6 × 10.sup.-4                                                                   6.4 × 10.sup.-5                           10.sup.-6  10.sup.-4                                                    67    7.4 ± 0.9 ×                                                                     5.5 ± 0.5 ×                                                                     2 × 10.sup.-4                                                                   6.5 × 10.sup.-5                           10.sup.-5  10.sup.-5                                                    68    2.6 ± 0.2 ×                                                                     2.0 ± 0.2 ×                                                                     not tested                                                                            1.0 ×  10.sup.-4                          10.sup.-4  10.sup.-4                                                    69    5.0 ± 0.04 ×                                                                    3.7 ± 0.7 ×                                                                     3 × 10.sup.-4                                                                   6.0 × 10.sup.-5                           10.sup.-4  10.sup.-4                                                    ______________________________________                                         Table 4 gives the comparative activity of each compound as determined by      various methods of measuring superoxide scavenging. The values represent      the concentration of each compound at which 50% superoxide inhibition was     achieved ±SEM. These are the averages of 3 experiments each done in        duplicate.                                                                    (a) xanthinexanthine oxidase system.                                          (b) and (c) macrophage system, without or in the presence of .15% BSA as      chelating agent, respectively.                                                (d) Drug concentration at which 50% of protein synthesis in the cells is      inhibited.                                                               

III. Anti-ascites (tumor) activity of compound 23:

The effect of compound 23 on ascites tumor development in C57/B1 micewas tested. Each mouse was injected intraperitoneally with either 5×10⁵or 1×10⁶ Ehrlich ascites cells. Half of the mice received injections of12-16 mg/kg of compound 23 and half remained untreated. Body weight andsurvival were tested at the time intervals indicated in FIGS. 2 and 3.The results of two (out of seven) representative experiments aredepicted in FIGS. 2 and 3. The main conclusions so far are: a) thecompound inhibits tumor proliferation and delays mortality caused by thetumor very considerably; b) experiments with doses (toxicity) andintervals between injections are needed; and c) the results areencouraging because the amount of tumor cells injected is very high andkills 100% of the animals within 20-21 days.

IV. Burn/wound healing for Compounds 23 and 67:

Female and male Hartley derived albino guinea-pigs were used throughoutthis study. They were housed individually and fed ad libitum normalguinea-pig's chow and water, supplemented with 1 gm of Vitamin C perweek.

The back of each animal was clipped and depilated 24 hours prior to theburn injury. Two symmetrical mirror-image round burns were inflicted onthe back of each animal with an aluminum template heated to 75° C. andapplied for 5 seconds under general anaesthesia (ketamine 150 mg/kgI.M.). Intraortic india ink injection indicated in our preliminarystudies that the injuries were deep second-degree burns. The burn areaswere dressed in the same fashion as is routinely done in cases of suchburns.

Application of the compounds for Experiments 3, 4 and 5 was carried outas follows: five times a day for the first four days and twice dailyfrom the fifth to the 12th day. On day twelve the animals weresacrificed and the wound tissues were sent for histological examination.

Control wounds were treated with 1 mg bovine serum albumin/1 ml salineor with 1 ml of saline only. Catalase treatment was also applied in 1mg/ml saline. Compound 23 was applied at either 3 mg/ml or varyingconcentrations of 3 mg/ml for the first four days, 2 mg/ml twice dailyfrom the 5th to the 8th day and 1 mg/ml twice daily from the 9th to the12th day.

The wounds were selected for treatment at random. Three or fourtreatment groups were performed in each experiment. All dressings werechanged under general anaesthesia every four days, at which time woundtracing for healing analysis was performed.

The evaluation of healing is based upon the following four criteria:

1. Epithelialization--wound epithelialization expressed as percent wascalculated employing the following formula: ##EQU1## where E₁ =rate ofepithelialization expressed in percent.

A12total=wound size on the: 12th day post burn both opened and healedwound.

Ao=area of open wound on the same 12th day.

E1 represents the % of the newly covered area out of the total woundarea on the 12th day. E2 represents the % of the healed area on the 12thday out of the total area of the primary wound induced at zero time.

2. Contraction--The burn wound size as delineated by its outerboundaries was traced on a transparent polyethylene sheet on post burndays (PBD) 4, 8 and 12. Areas were measured by a system composed of anIBM PC computerized video-camera interfaced with a specially designedsoftware. The percentage of contraction was calculated according to thefollowing formula: ##EQU2## where: C--percentage of contraction on PBD12.

A₁ --initial burn wound size.

A₁₂ --burn wound size PBD 12 (opened and healed wound.

Excessive contraction leads to varying degrees of limitation of use ofhealed areas and is thus unfavorable.

3. Assessment of the newly formed granulation tissue--Since theevaluation of collagen synthesis is precluded in burn wound models, thenewly formed granulation tissue in each burn wound was assessedhistologically on PBD 12. Four adjacent sections were taken from thecenter of each wound. Employing the Mason's Trichrome staining methodaided to delineate the newly formed granulation tissue which originatedfrom the non-burned dermal layer in each section.

4. Hair follicle count--The preservation of hair follicles and theircount was quantified. The presence of hair follicles and theirregeneration indicated preservation of blood circulation and the degreeof healing of the underlying tissues of the dermis.

Results

1. Epithelializtion--Tables 5 and 6--is clearly superior in the woundsreceiving Compound 23 treatment being 50.1% to 81.4% for variousCompound 23 concentrations, as compared with the average of 36.2% of thecontrol wound (34.4% and 38.6% accordingly) which were treated witheither BSA or saline only. Catalase treatment prevented new epithelialformation almost completely--and most of the wound remained unhealed.

2. Contraction--is a natural process occurring in each healing wound andtoo much contraction during healing may cause organ dysfunction. Theresults in Tables 5 and 6 indicate that Compound 23, particularly in thehigher concentration, did not enhance contraction and may actuallysomewhat reduce this process.

3. Granulation tissue formation--In the five control wounds and threeCompound 23 treated wounds analyzed from experiment no. 3, the thicknessof the newly formed collagen in controls was 539 on the average whereasin Compound 23 treated wounds it was 467. This constitutes 86% of thecollagen formed in untreated healing indicating that Compound 23 doesnot cause and any reduce fibrosis. This means that the compound reducesthe amount of scar tissue and allows more normal tissue to be formed inthe treated wounds.

4. Hair Follicle Count--The presence of hair follicles indicatesmaintenance of dermal microcirculation and, prevention of tissueischemia and thus ischemic and postischemic damage. The preservation ofhair follicles and their count were undertaken; the number ofregenerated hair follicles were counted microscopically in tissuesections. The biological section was projected onto a video, coloredscreen, connected to a color camera. Hair follicles were counted in eachwound, in three to eight microscopic fields,, in each of 4-8histological sections. Each field was 2 mm long and 5 microns thick. Themean and standard error (SE) were calculated for each wound and for thewhole treatment group. See especially Tables 8, 9 and 10 which appearhereinafter.

                  TABLE 5                                                         ______________________________________                                        experiment no. 3 (10 animals - 20 wounds)                                               Epithelialization %                                                          E1        E2       Contraction %                                     ______________________________________                                        control    34.4        21.3     35.8                                          Compound 23                                                                              50.1        31.7     35.7                                          3 mg/ml                                                                       catalase    6.1         2.8     41.4                                          ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        experiment no. 4 (11 animals - 22 wounds)                                               Epithelialization %                                                          E1        E2       Contraction %                                     ______________________________________                                        control    38.6        22.5     40.1                                          Compound 23                                                                              81.4        51.6     37.5                                          3 mg/ml                                                                       Compound 23                                                                              61.3        33.6     42.1                                          3/2/1 mg                                                                      ______________________________________                                    

In the subsequent experiment no. 5, we used 20 guinea pigs and inflicted32 wounds. 14 control wounds were treated with saline. 9 wounds weretreated with Compound-64 (3.8 mg/ml). 9 wounds were treated withCompound-23 mg/ml.

Table 7 summarizes the results of this experiment, with respect to %epithelialization and % contraction.

                  TABLE 7                                                         ______________________________________                                        experiment no. 5                                                                      % Epithelialization = E1                                                                     % Contraction                                          ______________________________________                                        control   69.2*            44.4                                               Compound 64                                                                             97.3**           42.1                                               3.8 mg                                                                        Compound 23                                                                             91.3**           37.0                                               ______________________________________                                         *higher control values over previous experiments due to changed frequency     of application of dressings.                                                  **virtually total recovery                                               

With respect to epithelialization the two compounds 23 and 64 showconsiderable improvement as compared to untreated. Contraction wassignificantly changed by the treatments.

The following Tables 8, 9 and 10 each summarizes all the parameterswhich were assessed for experiments #3, #4, #5 respectively.

There was no substantially significant difference in the initial woundsize among the treatment groups of each experiment as should beexpected.

Contraction was significantly different amongst the various treatmentgroups in experiments #3 and #4 However, in experiment #5 contractionwas significantly lower in the Compound 23 treated group as compared tocontrols (Table 7).

Contraction is a natural process occurring in each healing wound.Excessive contraction may lead to varying degrees of organ 1 imitationand is thus unfavorable. Contraction with the various treatments givenand especially with compound 23 did not exceed control values and wasactually reduced in experiment #5. This indicates that the drug does notheal by producing excessive fibrosis. A fibrotic scar is less aestheticand limits the functionality of the healed area.

In experiment #3 (Table 8) epithelialization was only 6% in catalasetreated group, 34.4% for control-B5A treated wound and. 50% for compound23. Epithelialization with compound 23 treatment was superior to controltreated wounds, it was not statistically significant. Thus catalasetreatment was significantly worse than either the control or compound 23treatments.

In experiment #4 (Table 9) treatment with 3 mg of compound 23significantly improved. epithelialization to 89% as compared with 38.5%in control treated wounds. The use of decreasing amounts of compound 23(treatment C) also improved e. pithelialization, but to a lesser degree.

In experiment #5 (Table 10) the wound dressings were not changed on the4th day, but only on the 8th and 12th PBD The reduced dressing inexperiment #5 resulted in improved healing of the wounds of all groupscompared with experiments #3 and #4. Epithelialization with 3mg ofcompound 23 was 90.3%, meaning that wounds were almost completely healedon day 12. Another Co compound was introduced, compound 64, and it alsodramatically improved epithelialization to 97%.

Across the three experiments reported hereinabove, epithelializaton wassuperior in all the groups treated with Co compounds. This should beconsidered in light of the crucial role of epithelialization in theprocess of healing.

Hair follicle preservation and formation is crucial in the assessment ofwound healing since it also indicates the maintenance of dermalmicrocirculation and prevention of tissue ischemia.

Hair follicle preservation in experiment #3 (Table 8) was verysignificantly better with compound 23 than either catalase or controltreated groups, the amount of hairs per field microscopic was twice asmuch in the compound 23 treated group; 15.5 in compound 23 vs. 8.2 and9.2 in control and catalase treatments, respectively.

Experiment #4 (Table 9) exhibited the same phenomenon, 3 mg of compound23 and decreasing amounts of compound 23 were superior to the controlgroup, though treatment with a higher compound 23 concentration wassuperior to decreasing amounts of the same treatment, i.e. 15.4 and 13.1hair follicles per field of compound 23 treated groups respectively vs.6.9 hair follicles for control. Very surprisingly compound 64, whichproved to be an inducer of epithelialization, did not improve hairfollicle preservation over control values, and was significantly lowerthan treatment with compound 23.

Across the three experiments reported hereinabove compound 23 provedsuperior with respect to hair follicle preservation and this coincidedwith superior epithelialization, which represents healing on amacroscopic level. Hair follicle preservation was two-fold higher thancontrol or any other treatment suggesting that microcirculation injuryand ischemia resulting from superoxide radical production were at leastpartially prevented by the use of compound 23 as superoxide radicalscavenger.

The thickness of the newly formed granulation tissue on PBD 12 was notfully assessed but in both experiments 3 and 4 (Tables 8, 9) thecollagen formed in the compound 23 treated group was slightly thicker(10% to 15%) than control values a difference which was notstatistically significant. The fact that the layer of new collagen didnot exceed that of control by more than 10% to 15% taken together withthe fact that contraction was lower due to treatment indicates that amore aesthetic scar will result from our treatment with compound 23.

The data indicate that burn wound healing in this guinea pig model wassignificantly accelerated in the groups treated especially withCo-compounds, i.e. compounds 23 and 64. This improvement was accompaniedby a significant increase in epithelialization. Moreover, compound 23also demonstrated improved hair follicle preservation, a fact whichindicates that the microcirculation in the burn area was at leastpartially protected and therefore regenerated better during the healingprocess. The fact that contraction and new granulation tissue did notincrease by compound 23 indicates that the scar tissue will not becomeexcessively fibrotic and thus will result in a more aesthetic scar and amore functional healing organ.

                  TABLE 8                                                         ______________________________________                                        Assesment of burn wound healing by means of                                   contraction, epithelialization, hair follicle                                 preservation, and newly formed granulation tissue.                            exp. 3                                                                        Female guinea pigs = 10, wounds (n) = 20                                      Weight = 530 + 10 gr.                                                         Treatments:                                                                            A (n = 6) catalase                                                                             1 mg/ml/treatment                                            B (n = 7) BSA = control                                                                        1 mg/ml/treatment                                            C (n = 7) Co-BAE-23                                                                            3 mg/ml/treatment                                            Treatment                                                                                   B                                                                 A           BBA =      C                                                      Catalase    control    Co-BAE-23                                   ______________________________________                                        Initial wound                                                                            1249.13 ± 25.64                                                                        1283.4 ± 68.7                                                                         1267 ± 37.94                             size - mm.sup.2                                                               Contraction                                                                              41.47 ± 4.73                                                                           35.83 ± 4.04                                                                          35.71 ± 2.42                             PBD-12(%)                                                                     Epithelialization                                                                        6.05 ± 6.05                                                                            34.4 ± 11.50                                                                          50.06 ± 12.14*                           PBD-12(%)                                                                     Hair follicles                                                                           9.25 ± 3.3                                                                             8.18 ± 2.15                                                                           15.5 ± 1.38**                            per 2 mm × 5                                                            Hair follicles                                                                           113.4       100        189**                                       % of control                                                                  New collagen -                                                                           not         654.6 ± 16.0                                                                          730.72 - 44.6                                          assessed                                                           ______________________________________                                         * = statistically significant difference from control                         ** = very significant difference from control                            

                  TABLE 9                                                         ______________________________________                                        Assessment of burn wound healing, by means of                                 contraction, epithelialization, hair follicle                                 preservation, and newly formed granulation tissue.                            exp. 4                                                                        Male animals = 11, wounds (n) = 22                                            Weight = 602.7 ± 24 gr.                                                    Treat-                                                                              A (n = 8) Co-BAE-23                                                                          3 mg/ml/treatment                                        ments:                                                                              B (n = 7) control -                                                                          1 ml/treatment                                                 saline                                                                        C (n = 7) Co-BAE-23                                                                          3 mg/ml/treatment                                                                           1-4 PBD                                                         2 mg/ml/treatment                                                                           5-8 PBD                                                         1 mg/ml/treatment                                                                           9-12 PBD                                            Treatment                                                                       A          B          C                                                       Co-BAE-23  Saline     Co-BAE-23                                               3 mg       control    3/2/1 mg                                     ______________________________________                                        Initial wound                                                                            1466.23 ±                                                                             1435.74 ±                                                                             1566.13 ± 105                             size - mm.sup.2                                                                          77.08      53.19                                                   Contraction                                                                              36.14 ± 2.89                                                                          40.08 ± 1.88                                                                          42.13 ± 2.94                              PBD-12(%)                                                                     Epithelialization                                                                        89.25 ± 38.55 ± 11.60                                                                         52.56 ± 16.21*                            PBD-12(%)  5.38**                                                             Hair follicles                                                                           15.4 ± 0.9**                                                                          6.9 ± 2.06                                                                            13.1 ± 2.72*                              per 2 mm × 5                                                            New collagen -                                                                           616.6 ± 46                                                                            576.5 ± 22.3                                                                          675.3 ±  79                               ______________________________________                                         * = some statistical significance from control                                ** = very significant difference from control                            

                  TABLE 10                                                        ______________________________________                                        Assessment of burn wound healing, by means                                    of contraction, epithelialization, hair follicle                              preservation, and newly formed granulation tissue.                            exp. 5                                                                        Female animals = 20, wounds (n) = 32                                          Weight = 532.5 + 6.02 gr.                                                     Treatments:                                                                           A (n = 9) Co-BAE-64                                                                            3.0   mg/ml/treatment                                        B (n = 14) control - saline                                                                    1     ml/treatment                                           C (n = 9) Co-BAE-23                                                                            3     mg/ml/treatment                                         Treatment                                                                       A           B          C                                                      BBAE-64     Saline control                                                                           Co-BAE-23                                   ______________________________________                                        Initial wound                                                                            1384.76 ± 35.7                                                                         1398 ± 34.1                                                                           1294.0 ± 33.9                            size - mm.sup.2                                                               Contraction                                                                              42.03 ± 1.89                                                                           44.89 ± 3.01                                                                          38.4 ± 3.17**                            PBD-12(%)                                                                     Epithelialization                                                                        97.29 ± 1.51**                                                                         69.25 ± 4.5                                                                           90.3 ± 5.33**                            PBD-12(%)                                                                     Hair follicles                                                                           9.9 ± 0.83**                                                                           10.6 ± 1.25                                                                           15.3 ± 2.39**                            per 2 mm × 5                                                            Hair follicles                                                                           93.4        100        144**                                       % of control                                                                  New collagen -                                                                           not         not        not                                                    assessed    assessed   assessed                                    ______________________________________                                                 Treatment                                                                       A           B          C                                                      C-23        C-67       Saline                                      ______________________________________                                        Initial wound                                                                            1171.84 ± 132                                                                          1117.6 ± 58.1                                                                         1107.37 ±                                size - mm.sup.2                   125.23                                      Contraction                                                                              29.16 ± 11.09                                                                          26.23 ± 9.42                                                                          29.07 ± 6.8                              PDN-16%                                                                       Epithelialization                                                                        57.5 ± 29.4                                                                            69.17 ± 12                                                                            57.4 ±                                   PBD-16(%) (E1)                    18.98*(a)                                   Epithelialization                                                                        38.98 ± 31.74                                                                          50.81 ± 11.21                                                                         41.8 ±                                   PBD-16(5) (E2)                    12.05*(b)                                   ______________________________________                                         Epithelialization (E1)  represents the % of the newly covered area of the     wound surface on the 16th post burn day out of the total wound area on th     same day.                                                                     Epithelialization (E2)  represents the % of the newly covered area of the     wound surface on the 16th post burn day out of the initial wound area on      the first day.                                                                ** = very significant difference                                              *marginally statistically significant from control.                      

The following examples are directed to the preparation of pharmaceuticalcomposition for the typical treatment of burns.

EXAMPLE I

An aerosol composition is prepared having the following proportions:

    ______________________________________                                        Benxocaine       1.00%                                                        Camphor          0.10                                                         Menthol          0.10                                                         Pyrilamine Maleate                                                                             0.25                                                         Bacitracin       0.02                                                         Acetulan.sup.R   1.00                                                         (Acetylated landin                                                            alcohols)                                                                     Oleyl Alcohol    4.00                                                         Dipropylene Glycol                                                                             1.00                                                         Compound 23      0.3                                                          Propellant 152 a/II                                                                            92.23                                                                         100.00%                                                      ______________________________________                                    

EXAMPLE II

The following water-soluble ointment is prepared having the followingproportions:

    ______________________________________                                        Polyethylene Glycol 200                                                       ______________________________________                                        Monostearate       15.0%                                                      Veegum             5.0                                                        Polysorbate 80     1.0                                                        Methylparaben      0.1                                                        Compound 23        0.3                                                        Purified water     78.6                                                                          100.00%                                                    ______________________________________                                    

EXAMPLE III

The following oleaginous ointment is prepared having the followingproportions:

    ______________________________________                                        Compound 64          .38%                                                     Petrolatum balance to 100%                                                    ______________________________________                                    

V. Evaluation of the potential of compound 23 in the presence of humanserum:

Material and Methods

The microorganisms, growth conditions, MIC and MBC tests were performedin the same conditions as in the first parts. But instead of using BHand TSB as growth media sterile, human serum was used. Themicroorganisms were incubated in human serum with differentconcentrations of compound 23, for establishing the MIC, while the MBCwas determined on TSA as before.

Results and Discussion

The results summarized in Table 12 indicate that:

1) The gram (-) microoganisms show the same pattern as if they weregrown in the rich medium (BH), the MBC is 4 mg/ml approximately.

2) The gram (+) microorganisms grown in the serum are as sensitive asthose grown in BH except for Staph. Coagulase positive which is lesssensitive in sterum (MBC+3.2 mg/ml). The reason may be that thismicroorganism is able to coagulate the serum and thereby influence themode of action of compound 23.

                                      TABLE 12                                    __________________________________________________________________________    Sensitivity of gram positive and gram negative bacteria to C.sub.23           compound grown in human serum.                                                __________________________________________________________________________            Strep. β                                                                      Strep. α                                                   Microorganism                                                                         hemolytic                                                                          hemolytic                                                                          Enterococci                                                                          Staph. coagulase (+)                                                                    Staph. coagulase (-)                       C.sub.23 (mg/ml)                                                                      MBC* MBC  MBC    MBC       MBC                                        __________________________________________________________________________    0.4     5.10.sup.4                                                                         3.10.sup.4                                                                         4.10.sup.3                                                                           5.10.sup.4                                                                              1.10.sup.4                                 0.8     1.10.sup.4                                                                         5.10.sup.2                                                                         4.10.sup.2                                                                           1.10.sup.4                                                                              1.10.sup.3                                 1.6     8.10.sup.3                                                                         1.10.sup.2                                                                         2.10.sup.2                                                                           1.10.sup.3                                                                              5.10.sup.2                                 3.2     5.10.sup.3                                                                         5.10.sup.1                                                                         2.10.sup.1                                                                           1.10.sup.2                                                                              2.10.sup.2                                 6.4     1.10.sup.2                                                                         1.10.sup.1                                                                         1.10.sup.1                                                                           1.10.sup.1                                                                              1.10.sup.1                                 __________________________________________________________________________    Microorganism                                                                          E. coli                                                                            Klebsiella                                                                            Pseudomonas                                                                           Proteus                                                                              C. albicans                              C.sub.23 (mg/ml)                                                                      MBC   MBC     MBC     MBC    MBC                                      __________________________________________________________________________    2       8.10.sup.3                                                                          8.10.sup.3                                                                            8.10.sup.3                                                                            3.10.sup.3                                                                           3.10.sup.3                               4       8.10.sup.2                                                                          2.10.sup.2                                                                            7.10.sup.3                                                                            4.10.sup.2                                                                           1.10.sup.3                               8       6.10.sup.1                                                                          3.10.sup.1                                                                            1.10.sup.3                                                                            8.10.sup.1                                                                           1.10.sup.2                               16      0     0       1.10.sup.2                                                                            1.10.sup.1                                                                           1.10.sup.2                               __________________________________________________________________________      *MBC = Minimal Bactericidal Concentration.                                   The initial concentration of microorganisms was 5.10.sup.4 /ml.          

VI. Comparison of the sensitivity of ten different strains of clinicalisolates of Psedomonas and the strain of Staph. coagulase positive tocompound

23:

The purpose of these experiments was to compare ten strains ofPseudomonas and Staph. (+) which showed various patterns of sensitivityto antibiotics, to their sensitivity to compound 23 compositions.

Material and Methods

Microorganisms--Ten clinical isolates of Pseudomonas aeruginosa and tenof Staph. (+) with various patterns of sensitivity to antibiotics weretested.

Growth conditions--As mentioned in the first part, but were tested onlyon TSB medium.

MIC and MBC--as described above.

Results and Discussion

Although many of the strains tested (Pseudomonas as well as Staph.coagulase positive) were very resistant to many antibiotics tested (asGentamcin, and all the new cephalosporins), their sensititivity tocompound 23 compositions was very similar (Table 13). Compound 23 forPseudomonas was bactericidal at 4 mg/ml, whereas for Staph. (+) it wasbactericidal 1.5 mg/ml-3.0 mg/ml.

                                      TABLE 13                                    __________________________________________________________________________    Sensitivity to C-23 of various strains of clinical isolates                   __________________________________________________________________________    (a) Pseudomonas                                                               Microorganism                                                                         Ps.(1)  Ps.(2) Ps.(3) Ps.(4) Ps.(5)                                   C.sub.23 mg/ml                                                                        MIC.sup.1                                                                         MBC.sup.2                                                                         MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC                                   __________________________________________________________________________    2       -   4.10.sup.4                                                                        -  5.10.sup.4                                                                        -  4.10.sup.4                                                                        -  3.10.sup.4                                                                        -  5.10.sup.4                            4       -   1.10.sup.3                                                                        -  4.10.sup.4                                                                        -  1.10.sup.2                                                                        -  1.10.sup.3                                                                        -  1.10.sup.3                            8       -   2.10.sup.2                                                                        -  1.10.sup.1                                                                        -  0   -  0   -  0                                     16      -   0   -  1.10.sup.1                                                                        -  0   -  0   -  0                                     __________________________________________________________________________    Microorganism                                                                         Ps.(6)  Ps.(7) Ps.(8) Ps.(9) Ps.(10)                                  C.sub.23 mg/ml                                                                        MIC MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC                                   __________________________________________________________________________    2       -   1.10.sup.4                                                                        -   5.10.sup.4                                                                       -  2.10.sup.4                                                                        +  1.10.sup.4                                                                        +  2.10.sup.4                            4       -   0   -  2.10.sup.3                                                                        -  6.10.sup.3                                                                        -  0   -  8.10.sup.1                            8       -   0   -  0   -  0   -  0   -  0                                     16      -   0   -  0   -  0   -  0   -  0                                     __________________________________________________________________________    (b) Staph. coagulase positive                                                         Staph(+) 1                                                                            2      3      4      5                                                MIC MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC                                   __________________________________________________________________________    0.2     +       +      +      +      +                                        0.4     +       +      +      +      +                                        0.75    +       -  8.10.sup.3                                                                        +      +      -  1.10.sup.4                            1.5     -   1.10.sup.4                                                                        -  0   -  4.10.sup.4                                                                        -  4.10.sup.1                                                                        -  6.10.sup.1                            3       -   0   -  0   -  6.10.sup. 2                                                                       -  0   -  0                                     __________________________________________________________________________            6       7      8      9      10                                               MIC MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC                                   __________________________________________________________________________    0.2     +       -  3.10.sup.4                                                                        -  2.10.sup.4                                                                        -  8.10.sup.4                                                                        -  5.10.sup.4                            0.4     +       -  1.10.sup.4                                                                        -  8.10.sup.3                                                                        -  1.10.sup.4                                                                        -  9.10.sup.3                            0.75    -   2.10.sup.4                                                                        -  1.10.sup.4                                                                        -  6.10.sup.3                                                                        -  1.10.sup.4                                                                        -  9.10.sup.3                            1.5     -   8.10.sup.3                                                                        -  8.10.sup.3                                                                        -  0   -  1.10.sup.4                                                                        -  3.10.sup.3                            3       -   1.10.sup.2                                                                        -  1.10.sup.2                                                                        -  0      8.10.sup.3                                                                        -  1.10.sup.2                            __________________________________________________________________________     1. MIC + Minimal Inhibitory Concentration. (=) visible/growth; (-)            transparent.                                                                  2. MBC = Minimal Bactericidal Concentration.                                  The initial concentration of microorganisms was 5.10.sup.4 /ml.          

Sensitivity of various microorganisms were tested to:

(a) Silver sulfadiazine (SSD)

(b) Sulfamylon (SM)

(c) Compound 23.

The purpose of this study was to compare the sensitivity of SSD and SM,which are well known antimicrobial agents, to compound 23.

The microorganisms, growth conditions, MIC and MBC tests are performedas described in the first part, on TSB medium.

Results and Discussion

Silver Sulfadiazine is effective against the strains tested (grampositive and negative as well) at low concentration (bactericidal at 50μg/ml) in comparison with compound 23 (Table 14).

But Sulfamylon is effective at low concentrations (75-150 μg/ml) onlyfor the Streptococci, whereas for the other gram (+) bacteria tested(namely, Staphylococci or Enterococci) or gram. (-) bacteria very highconcentrations are needed to be used to get a bactericidal effect(12,000 μg/ml). This concentration is much higher than that of compound23. C. albicans is not sensitive even at that concentration.

                                      TABLE 14                                    __________________________________________________________________________    Sensitivity of gram positive and gram negative bacteria to Silver Sulfa       Diazine SSD                                                                   and Sulfamylon SM.                                                            __________________________________________________________________________    Microorganism                                                                         Strep. β                                                                         Strep. α                                                                       Enterococci                                                                          Staph (+)                                                                            Staph (-)                                SSD μg/ml                                                                          MIC.sup.1                                                                         MBC.sup.2                                                                         MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC                                   __________________________________________________________________________    25      -   7.10.sup.4                                                                        -  7.10.sup.4                                                                        +      +      +                                        50      -   7.10.sup.3                                                                        -  8.10.sup.1                                                                        +      -  7.10.sup.3                                                                        -  5.10.sup.4                            75      -   0   -  0   -  7.10.sup.4                                                                        -  1.10.sup.3                                                                        -  1.10.sup.3                            100                                                                           150                                                                           __________________________________________________________________________    Microorganism                                                                          E. coli Klebsiella                                                                             Pseudomonas                                                                            Proteus                                    SSD μg/ml                                                                          MIC MBC  MIC MBC  MIC MBC  MIC  MBC                                   __________________________________________________________________________    25                                                                            50      -   8.10.sup.3                                                                         -   1.10.sup.3                                                                         -   5.10.sup.4                                                                         -    0                                     75      -   2.10.sup.2                                                                         -   0    -   3.10.sup.3                                                                         -    0                                     100     -   0    -   0    -   3.10.sup.1                                                                         -    0                                     150         0        0        0         0                                     __________________________________________________________________________    Microorganism                                                                         Strep. β                                                                         Strep. α                                                                       Enterococci                                                                          Staph (+)                                                                            Staph (-)                                SM μg/ml                                                                           MIC.sup.1                                                                         MBC.sup.2                                                                         MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC MIC                                                                              MBC                                   __________________________________________________________________________     75     -   2.10.sup.3                                                                        -                                                             150     -   9.10.sup.2                                                                        -  1.10.sup.3                                                 300     -   6.10.sup.2                                                                        -  1.10.sup.3                                                 600     -   6.10.sup.2                                                                        -  1.10.sup.3                                                 3000                   +  1.10.sup.4                                                                        -  1.10.sup.4                                                                        -  1.10.sup.4                            6000                   -  1.10.sup.4                                                                        -  1.10.sup.4                                                                        -  1.10.sup.4                            12,000                 -  5.10.sup.3                                                                        -  5.10.sup.3                                                                        -  3.10.sup.3                            __________________________________________________________________________    Microorganism                                                                          E. coli Klebsiella                                                                             Pseudomonas                                                                            Proteus                                    SSD μg/ml                                                                          MIC MBC  MIC  MBC MIC MBC  MIC  MBC                                   __________________________________________________________________________     75                                                                           150                                                                           300                                                                           600                                                                           3000    +        +        +        +                                          6000    -        -        -        +                                          12,000  -   1.10.sup.4                                                                         -   1.10.sup.4                                                                         -   1.10.sup.4                                                                         -    1.10.sup.4                            __________________________________________________________________________     1. MIC = Minimal Inhibitory Concentration. (+) visible growth; (-)            transparent.                                                                  2. MBC = Minimal Bactericidal Concentration.                                  The initial concentration of microorganism was 5 × 10.sup.4 /mL    

VII. Mutagenicity of compounds #23 and 64:

Test was carried out on Photobacterium fisherii as described in Methodsin Enzymology Vol. 133:264-284(1986) by S. Ulizur who also carried outthe present tests. This test is more sensitive than the Ames test (seereference).

    ______________________________________                                        Conc. in                                                                              #23             #64                                                   mg/ml   growth   luminescence                                                                             growth luminescence                               ______________________________________                                        10      -        0          -      0                                          5       -        0          -      0                                          2.5     -        0          -      0                                          1.25    -        0          -      0                                          0.6     -        0          -      0                                          0.3     -        0          -      0                                          0.15    -        0          -      7                                          0.075   -        0.5        +      130                                        0.037   ++       500        ++     300                                        control ++       800        ++     800                                        ______________________________________                                    

Conclusion: These compounds are efficient antimicrobials to thisgram-negative bacterium. No mutagenic activity is revealed by backmutation to luminescent state.

The values are given as luminescence (quanta/sec⁻¹ /ml⁻¹). Significantgenotoxic effect of a compound is considered when the maximalluminescence developed due to the chemical in question is 3-4 timeshigher than that obtained with the control.

VIII. Activity of Compounds Outside Defined Class:

The following compounds were found to be unreactive, meaning they showno scavenging activity below concentrations of about 10⁻² M:

Co(en)₃ Cl ₃. 3H₂ O (where en=ethylenediamine);

Co(pn)₃ Cl₃ (where pn=propylenediamine);

[Co(Tim-6,13-(OH)₂)Br₂ ]Br (the compound is shown below and is alsotoxic); ##STR23## Ferrocene and Ferrocene derivatives (very effective asO₂ ⁻ scavengers but has absolutely no biological activity. In burnwounds, these compounds are worse than the controls).

REFERENCES

1. G. Costa, G. Mestroni. G. Tauzer and L. Stefani, Journal ofOrganometallic Chemistry, 1966, 6, 181-187.

2. J. P. McCarthy, R. J. Hovey, K. Ueno, A. E. Martell, J.A.C.S., 1955,77, 5820-5824.

3. K. Kasuga, T. Nagahara, A. Tsuge, K. Sogabe, Y. Yamamoto, Bull. Chem.Soc. Jpn, 1983, 56, 95-98.

4. T. Nagahara, K. Kasuga and Y. Yamamoto, Inorg. Nucl. Chem. Letters,1981. 17, 7-8, 235; k. Kasuga, Y. Iida, Y. Yamamoto, M. Aihara and M.Kudo, Inorganica Chimica Acta, 1984, 84, 113; T. Nagahara, K. Kasuga andY. Yamamoto, Inorgani ca Cimica Acta, 1981, 47, 37.

4. A. W. Johnson, E. Markham and R. Price, Org. Synth., Collectivevolume V, 785.

6. W. Dieckman, Chem. Ber., 1912, 2689.

7. R. S. Drago and B ,. R. Corden, Acc. Che. Res., 1980, 13, 353.

8. E. C. Niederhoffer, J. H. Timmons and A. E. Martell, Chem. Rev.,1984, 84, 137.

9. A. Summerville, R. D. Jones, B. M. Hoffman and F. Basolo, J. Chem.Educ., 1979, 56, 3, 157.

10. D. Getz, E. Melamud, B. L. Silver and Z. Dori, J. Am. Chem. Soc.,1975, 97, 3846.

What is claimed is:
 1. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a complex in an amount effectiveto alleviate undesirable symptoms associated with the presence of freeradicals, the complex comprising a Co(Ill) complex having an octahedralbasal plane defined by four donor atoms A, which may be same ordifferent, and two axial ligand donor atoms B, which may be the same ordifferent, said donor atoms having a low to intermediate ligand fieldstrength, said complex reacting with O₂ - to form a Co(Ill)-O₂ adduct oroxidizing O₂ - to produce dioxygen and a Co(II) complex.
 2. Thepharmaceutical composition of claim 1 wherein the complex has aquadridentate ligand L bound to the Co(III) through the donor atomswhich imposes planarity on the octahedral basal plane.
 3. Thepharmaceutical composition of claim 2 wherein the quadridentate ligand Land bonded Co(III) comprises a 6,5,6 ring system, said 6-membered ringof said 6,5,6 ring system being unsaturated.
 4. The pharmaceuticalcomposition of claim 3 wherein the complex has the formula [CoL(B)₂ ]²wherein B is selected from the group consisting of I⁻, Br⁻, Cl⁻, F⁻ OH⁻,C₂ O₄ ²⁻, H₂ O, and NH₃, and n is -1, 0, or +1.